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| United States Patent |
5,225,534
|
|
Certa
|
July 6, 1993
|
Recombinant malarial polypeptides
Abstract
The invention provides polypeptides which correspond in at least one
specific epitope with a plasmodium falciparum merozoite antigen having a
molecular weight of about 41,000 Daltons, and a process for their
production. The invention further provides immunogenic compositions which
contain such a polypeptide and a suitable adjuvant, a DNA sequence which
codes for such a polypeptide, replicable microbial vectors which contain
such a DNA sequence, microorganisms which contain such a replicable vector
and antibodies against a polypeptide of the invention. The invention still
further provides processes for the production of the immunogenic
compositions, the microorganisms and the antibodies and for the use of the
polypeptides and the immunogenic compositions for the immunization of
mammals against malaria.
| Inventors:
|
Certa; Ulrich (Allschwil, CH)
|
| Assignee:
|
Hoffmann-La Roche Inc. (Nutley, NJ)
|
| Appl. No.:
|
737126 |
| Filed:
|
July 29, 1991 |
Foreign Application Priority Data
| Current U.S. Class: |
530/350; 424/191.1; 424/268.1; 530/300 |
| Intern'l Class: |
C07K 013/00; C07K 007/10; A61K 039/00 |
| Field of Search: |
424/88
530/350,300
|
References Cited
| Foreign Patent Documents |
| 75245/87 | Jun., 1987 | AU.
| |
| 283829 | Mar., 1988 | EP.
| |
| 88/1775 | Mar., 1988 | ZA.
| |
| 86/04922 | Aug., 1986 | WO.
| |
Other References
Siddiqui et al. I & I 52:314-318 1986.
Del Guidice et al. J. of Imm. 137: 2952-2955 1986.
Perrin et al. J. Clin. Invest. 75:1718-172 1985 Immunization with a
Plasmodium Falciparum Merozoite Surface Antigen Induces Partial Immunity
in Monkeys.
Weber, et al., Nucleic Acids Res. 14:3311-3323 (1986).
Cheung, et al., EMBO J. 4:1007-1011 (1985).
McGarvey, et al., Proc. Natl. Acad. Sci. USA 81:3690-94 (1984).
|
Primary Examiner: Nucker; Christine M.
Assistant Examiner: Sidberry; H.
Attorney, Agent or Firm: Gould; George M., Epstein; William H., Roseman; Catherine R.
Parent Case Text
This is a division of application Ser. No. 07/237,126, filed Aug. 29, 1988,
now U.S. Pat. No. 5,061,788, issued Oct. 29, 1990.
Claims
What is claimed is:
1. A polypeptide having the amino acid sequence
##STR3##
wherein --W-- is Gln or can be absent;
--X-- is Met or Gin;
--Y-- is Gly or Cys and
--Z-- is Gly or Cys
which is covalently linked with an affinity peptide.
2. A polypeptide having the amino acid sequence
##STR4##
wherein --W-- is Gln or can be absent;
--X-- is Met or Gin;
--Y-- is Gly or Cys and
--Z-- is Gly or Cys, and
which is absorbed on or covalently coupled to a carrier material.
Description
TECHNICAL FIELD
This invention relates to recombinant malarial polypeptides having epitopes
of the plasmodium falciparum merozoite antigen, and to such polypeptides
which are covalently linked to affinity peptides.
BACKGROUND OF THE INVENTION
Malaria in human beings is caused by four species of plasmodium, P.
falciparum, P. vivax, P. ovale and P. malariae. According to a 1986 report
of the World Health Organization (WHO), there are almost 100 million cases
of malaria infection worldwide. Of these about 1 million, mostly cases of
young children who are infected with p. falciparum, are fatal. Because of
the appearance of drug resistant parasites and insecticide resistant
mosquito vectors, malaria is spreading. Thus, the Indian Health
Authorities reported 100,000 cases of malaria in 1962 and 3 million cases,
caused mainly by p. vivax, in 1980 (see Bruce-Chwatt, Essential
Malariology, 2nd edition, Heinemann, London [1986]).
Recent technical advances have raised hopes that it would soon be possible
to produce an antimalarial vaccine which would counteract the growing
spread of the disease. Firstly, new methods in the development of malarial
vaccines, e.g., the cloning of genes and their expression in microbial
host organisms and the use of monoclonal antibodies for antigen
identification, can be used. Secondly, long-term cultures of p. falciparum
in human red blood cells (Trager et al., Science 193, 673-675 [1976]) have
provided a ready source of material for the study of the malaria parasite.
More recently, it has become possible to maintain all stages in the life
cycle of the parasite in the laboratory (Ponnudurai et al., Trans. R. Soc.
Trop. Med. Hyg. 76, 812-818[1982]; Mazier et al., Science 227, 440-442
[1985]).
The natural life cycle of P. falciparum has three different stages. In the
first stage, mosquitoes introduce sporozoites into the blood vessels of
vertebrates during the intake of food. These sporozoites travel via the
bloodstream to the liver and invade the hepatocytes of the host. In the
second stage, merozoites develop from these sporozoites. These merozoites
pass through several multiplication cycles in erythrocytes of the host and
then develop to gametocytes. The gametocytes, which are the sexual stage
of the parasite, are taken up by mosquitoes when they feed. After
fertilization in the stomach of the insect, the gametocytes develop into
sporozoites which then travel to the salivary glands of the insect. There,
the cycle can begin again.
Sporozoites, merozoites and gametocytes have different antigens. Vaccines
can be produced in principle against any of the different stages of the
malaria parasire, but it is known that many polypeptides of the parasite
are genetically polymorphic, i.e. that the polypeptide changes slightly
from generation to generation. This hinders the immunization of
vertebrates against malaria using these polypeptides as antigens, since
the once-formed antibodies in time can no longer recognize the altered
antigens. Accordingly, an ideal vaccine would be one which is directed
against a polypeptide of the parasite having an amino acid sequence which
is not variable, i.e. against a polypeptide which is genetically stable.
It is known that the amino acid sequence (primary structure) of
polypeptides which carry out a specific function, such as enzymes, is
constant at least in those regions of the primary structure which are
important for function.
An example of a genetically stable polypeptide of P. falciparum is the
merozoite antigen having the amino acid sequence (I):
##STR1##
wherein -W- is Gln or can be absent;
-X- is Met Gln;
-Y- is Gl Cys and
-Z- is Cys.
BRIEF DESCRIPTION OF THE FIGURES
The following figures and the detailed example below will facilitate better
understanding of the present invention. However, the invention is not
limited by the Example or by the Figures, which are offered by way of
illustration only.
B, Bg, E, H, Sa, X and Xb denote cleavage sites for the restriction enzymes
BamHI, BglII, EcoRI, HindIII, SalI, XhoI and XbaI, respectively.
represents the promoters of the genes bla. lacI and neo; represents
the ribosomal binding sites of rhe genes bla, cat, neo and lacI;
represents the terminators t.sub.o and Tl; represents the regulatable
promoter/operator element N250pSN250p29; represents the ribosomal
binding site RBSII; .fwdarw. rep the coding region under control of this
ribosomal binding site; represents a region which codes for the six
histidines; represents the region which is required for replication
(repl.); represents coding regions for dihydrofolate reductase (dhfr),
chloramphenicol acetyltransferase (cat), lac repressor (lacI),
.beta.-lactamase (bla) and neomycin phosphotransferase (neo).
FIG. 1 Schematic representation of the plasmid pDS7B/RBSII.
FIG. 2 (FIG. 2a, FIG. 2b, FIG. 2c, and FIG. 2d) Nucleotide sequence of the
plasmid pDS78/RBS In the seguence the recognition sites for the
restriction enzymes set forth in FIG. 1 are overlined, while the regions
coding for .beta.-lactamase and dihydrofolate reductase are underlined.
FIG. 3 Schematic representation of the plasmid pDMI, 1.
FIG. 4 (FIG. 4a, FIG. 4b, and FIG. 4c) Nucleotide sequence of the plasmid
pDMI,l. In the sequence the recognition sites for the restriction enzymes
set forth in FIG. 3 are overlined, while the regions coding for neomycin
phosphotransferase and lac repressor are underlined.
FIG. 5 Schematic representation of the production of the XhoI/BamHI
fragment having the regulatable promoter/operator element N250PSN250P29,
the ribosomal binding site RBSII and the region coding for six histidines.
FIG. 6 Schematic representation of the construction of the plasmid
pDS78/RBSII.6xHis using the plasmids pDS78/RBSII and the XhoI/BamHI
fragment F having the regulatable promoter/operator element N250PSN250P29,
the ribosomal binding site RBSI and the region coding for six histidines.
FIG. 7 Southern Transfer Analysis (Southern. J. Mol. Biol. 98. 503-517
[1975]of genomic DNA from 12 different strains of p. falciparum. All 12
isolates have the typical DraI fragments. The P. falciparum DNA fragment
from Kl-B served as the probe.
FIG. 8 Western Transfer Analysis (Western blot, Towbin et al., proc. Natl.
Acad. Sci. USA, 76, 4350-4354 [1979]) of p. falciparum proteins from 11
isolates having antibodies against the merozoite antigen of p. falciparum.
All 11 isolates have the characteristic bands which correspond to a
polypeptide having a molecular weight of about 41,000.
FIG. 9 Expression of the recombinant protein (27 kD) in E. coli.
E. coli cell lysates were tested in the Western blot with antibodies
against the parasite antigen. The trace denoted as MW-ST contains
pre-stained molecular weight standard. The size is given in kilodaltons
(1,000 Daltons). Trace 1 contains a non-induced lysate of the transformed
cells. Trace 2 contains an induced probe. Trace 3 contains non-transformed
cells as rhe control. Trace 4 contains a p. falciparum Kl lysate. As
expected, the antibodies react only with the recombinant protein (27 kD)
in Trace 2 and with the parasite protein (41 kD) in Trace 4.
FIG. 10 purification of the recombinant protein (41 kD).
Analytical polyacrylamide gel electrophoreses and Western blot analysis of
the various purification steps in the purification of the recombinant
protein, (41 kD).
(10A) polyacrylamide gel stained with Coomassie blue. Trace 1: Cell lysate
of E. coli cells transformed with p8/3. Trace 2; Soluble fraction of the
cell lysate after centrifugation (100,000 .times.g). Trace 3: Eluate of
the material bonded specifically to the phosphocellulose column. Trace 4:
Eluate of the material bonded specifically to the NTA resin. Trace 5: End
product after ultrafiltration on a Sephacryl.TM. S-200 column. The
following molecular weight marker proteins were used:
31=carboanhydrase molecular weight
(MW)=31,000 Dalton. 45=ovalbumin MW=45,000
Dalton, 66=bovine serum albumin MW=66, 000
Dalton, 92=phosphorylase B MW=92,000 Dalton.
(10B) Western blot of the polyacrylamide gel (A) with rabbit antiserum
against an E. coli lysate.
(10C) Western blot of the polyacrylamide gel (10A) with antibodies against
merozoite antigens of P. falciparum (perrin et al., J. Clin. Invest. 75,
1718-1721[1985]).
FIG. 11 (FIG. 11a, FIG. 11b, FIG. 11c, FIG. 11d, and FIG. 11e) Nucleotide
sequence of the plasmid p8/3.
The sequence which codes for the polypeptide (41 kD) begins with the ATG at
position 115-117 (S) and ends with the stop codon at position 1255-1257
(T). The sequence which codes for the affinity peptide begins with the
aforementioned ATG and ends with the tyrosine (Tyr) coding codon TAT at
position 166-168, while the sequence coding for the partial sequence B of
the polypeptide (41 kD) begins with the codon ATG at position 169-171
which codes for methionine (Met) and likewise ends with the stop codon at
position 1255-1257.
FIG. 12 Nucleotide sequence and the amino acid sequence derived therefrom
of the genomic DNA of the p. falciparum Kl isolate which codes for the
41,000 Dalton merozoite antigen. The N-terminal Met is underlined. The
open reading frame ends with the termination codon TAA at position 1087 to
1089. The Figure also shows a part of the non-coding seguence prior to and
after the sequence coding for the merozoite antigen. The nucleotide
sequence of the genomic DNA of another isolate (RO-33, Ghana) was largely
identical with the nucleotide sequence from the Kl isolate. M25 denotes
the corresponding nucleotide sequence of a cDNA of the M25 isolate from p.
falciparum. The nucleotide sequences differ in the coding sequence in 3
codons which are framed in FIG. 12. The differences in the sequences lead
to two amino acid exchanges, with a Met or a Gly in the merozoite antigen
of the Kl isolate corresponding to a Gln or a Cys in the merozoite antigen
of the M25 isolate.
DESCRIPTION OF THE INVENTION
The present invention provides polypeptides which correspond in at least
one specific epitope with the plasmodium falciparum merozoite antigen
having the amino acid sequence (I). A specific epitope is an immunogenic
determinant on a polypeptide which is formed by a specific molecular
configuration of a partial sequence of the polypeptide. The invention also
provides polypeptides as defined above which, in addition, are covalently
linked to an affinity peptide.
Affinity peptides contain sequences of amino acid residues which bind
preferably to affinity chromatography carrier materials. Examples of such
affinity peptide residues are peptide residues which contain at least two
histidine residues. Such affinity peptide residues bind selectively to
nitrilotriacetic acid-nickel chelate resins (see, e.g., European patent
Application, publ. No. 253 303). polypeptides which contain such an
affinity peptide residue can be separated selectively from the remaining
polypeptides by means of such resins. The affinity peptide can be linked
either with the C-terminus or the N-terminus of the polypeptide defined
above, but the linkage is preferably with the N-terminus, especially when
the natural stop codon of the malaria antigen is utilized in the
expression of the polypeptide in accordance with the invention.
The preferred polypeptide in accordance with the present invention can be
represented by the general formula
A--B
wherein
A is an affinity peptide or can be absent,
B is a polypeptide which corresponds in at least one specific epitope with
the P. falciparum merozoite antigen having the amino acid sequence (I).
The most preferred polypeptides in accordance with the invention have the
amino acid sequence:
##STR2##
The invention also provides polypeptides of rhe general formula A-B having
an amino acid sequence derived from the amino acid sequences indicated
above by additions, deletions, insertions or amino acid substitutions
provided that these polypeptides are still capable of eliciting an immune
response against the merozoite stage of malaria parasites, preferably
against the merozoite antigen having the amino acid sequence (I) of P.
falciparum. The invention also provides DNA sequences which code for a
polypeptide of the invention, and replicable microbial vectors which
contain such DNA sequences, especially expression vectors, i.e. replicable
microbial vectors, in which a DNA seguence which codes for a polypeptide
of the invention is operatively linked to an expression control sequence
in such a way that the DNA sequence coding for the polypeptide can be
expressed. Moreover, the present invention provides microorganisms which
contain such a replicable vector or expression vector and a process for
their production. Furthermore, the present invention provides a process
for the production of the polypeptides and methods for their use for the
immunization of mammals against malaria.
The amino acid sequences of the polypeptides of the invention can differ
from the amino acid sequences given above by having certain amino acid
substitutions which have no influence on spatial structure or biological
activity. Examples of such amino acid substitutions are Ala/Ser, Val/Ile,
Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/phe,
Ala/pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and vice versa (see
Doolittle, in "The proteins", Eds. Neurath, H, and Hill, R.L., Academic
press, New York [1979]).
The polypeptides can be covalently bound to a oarrier material or can be
adsorbed thereon. Suitable carrier materials are natural or synthetic
polymeric compounds such as. e.g., copolymers of one or more amino acids
(e.g., polysine) or sugars (e.g., polysaccharides). Other suitable carrier
materials are natural polypeptides such as hemocyanins (e.g., KLH, or
"keyhole limpet hemocyanin"). serum proteins (e.g., gammaglobulin, serum
albumin) and toxoids (e.g., diphtheria or tetanus toxoid). Other suitable
carrier materials are known to those skilled in the art.
The covalent bonding of the polypeptides of the invention to the carrier
materials can be effected in a known manner, e.g., directly by the
formation of a peptide or ester bond between free carboxyl, amino or
hydroxyl groups of the polypeptide and the corresponding groups on the
carrier material or indirectly by using conventional, bifunctional
reagents such as m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) or
succinimidyl 4-(p-maleimidophenyl)butyrate (SMpB). These and other
bifunctional reagents are commercially obtainable, e.g., from pierce
Chemical Company, Rockford, Ill. Furthermore, C.sub.2-7 -dialkanals, such
as glutaraldehyde (Avrameas, Immunochem. 6, 43-52 [1969]), can be used.
The carrier materials to which the polypeptides are bonded can be separated
from non-bonded polypeptides and, if desired, from excess reagents by
known methods (e.g., by dialysis or column chromatography).
The peptides can be produced by conventional methods of peptide synthesis
in the liquid phase or, preferably, on the solid phase, such as the
methods of Merrifield (J. Am. Chem. Soc. 85, 2149-2154 [1963]) or by other
equivalent methods commonly used in the art.
Solid phase synthesis begins with the C-terminal amino acid of the peptide
to be synthesized, which is coupled in protected form to an appropriate
resin. The starting material can be produced by coupling an amino acid,
which is protected at the amino group, to a chloromethylated or a
hydroxymethylated resin via a benzyl ester bridge or via an amide bond to
a benzhydrylamine (BHA) resin, a methylbenzhydrylamine (MBHA) resin or a
benzyloxybenzyl alcohol resin. These resins are commercially obtainable
and their production and use are well-known.
General methods for the protection and removal of protecting groups from
amino acids, which can be used in this invention, are described in "The
peptides", Vol. 2 (edited by E. Gross and J. Meienhofer, Academic press,
New York, 1-284[1979]). protecting groups include, e.g., the
9-fluorenylmethyloxycarbonyl (Fmoc), the tertiary butyloxycarbonyl (Boc),
the benzyl (Bzl), the t-butyl (But), the 2-chlorobenzyloxycarbonyl
(2Cl-Z), the dichlorobenzyl (Dcb) and the 3,4-dimethylbenzyl (Dmb) group.
After removal of the .alpha.-amino protecting group, the protected amino
acids are coupled stepwise in the desired seguence to the C-terminal amino
acid bonded to the resin. The complete peptide can thus be synthesized. As
an alternative thereto, small peptides can be synthesized and then joined
together to give the desired peptide. Suitable coupling reagents are well
known in the art; dicyclohexylcarbodiimide (DCC) is especially preferred.
Each protected amino acid or peptide is added in excess to the solid phase
synthesis reaction vessel and the coupling reaction can be carried out in
dimethylformamide (DMF) or methylene chloride (CH.sub.2 CH.sub.2) or a
mixture of both. In cases of incomplete coupling, the coupling reaction is
repeated before the N-terminal .alpha.-amino protecting group is removed
for the purpose of coupling the next amino acid. The yield of each
coupling step can be followed, preferably according to the ninhydrin
method. The coupling reactions and the washing steps can be carried out
automatically.
Cleavage of the peptide from the carrier material can be achieved by
methods which are well known in peptide chemistry. e.g., by reaction with
hydrogen fluoride (HF) in the presence of p-cresol and dimethyl sulphide
for 1 hour at 0.degree. C. followed possibly by a second reaction with HF
in the presence of p-cresol for 2 hours at 0.degree. C. The cleavage of
the peptides from chloromethylated or hydroxymethylated carrier materials
gives peptides having a free C-terminus; the cleavage of peptides from
benzylhydrylamine or methylbenzylhydrylamine carriers gives peptides
having an amidated C-terminus.
Alternatively, the polypeptides of the invention can be produced using
recombinant DNA technology (Manniatis et al., in "Molecular Cloning--A
Laboratory Manual", Cold Spring Harbor Laboratory [1982]). For example, a
DNA fragment which codes for a polypeptide can be synthesized by
conventional chemical methods, e.g., by the phosphotriester method
described by Narang et al. in Meth. Enyzmol. 68, 90-108[1979], or by the
phosphodiester method (Brown et al., Meth. Enzymol. 68, 109-151 [1979]).
In both methods, long oligonucleotides are first synthesized and then
joined together in a predetermined way.
The nucleotide sequence of the DNA fragment can be identical to the
nucleotide sequence which codes for the natural polypeptide in plasmodium
parasites. Since the genetic code is degenerate, however, there are many
other sequences that can also code for the same polypeptide. The codons
selected can be adapted to the preferred codon usage of the host used to
express the gene coding for the recombinant polypeptide (Grosjean et al.,
Gene 18, 199-209 [1982]). Care must be taken that the DNA fragment used
does not contain partial sequences which make the construction or use of
the expression vector difficult, e.g., by introducing an undesired
restriction enzyme cleavage site or by preventing the expression o(the
polypeptide.
polypeptides of the general formula A-B can also be produced by isolating a
DNA fragment which codes for the partial sequence B from the genome of a
plasmodium isolate and expressing it in a host organism. The DNA fragment
which codes for the partial sequence B can be obtained by cleaving genomic
DNA of a plasmodium strain with one or more suitable restriction
endonucleases, e.g., EcoRl. Fragments with a length of 1.5 to
8.times.10.sup.3 base pairs isolated and inserted into a suitable vector,
e.g., into the .lambda. phage vector gtll (Young et al., proc. Natl. Acad.
Sci. USA 80, 1194-1198 [1983]) obtainable from the American Type Culture
Collection. 12301 Parklawn Drive, Rockville, Md., USA (ATCC No. 37194).
The recombinant phage DNA can be packaged in phages in vitro. The
thus-obtained phages are introduced into suitable host cells, e.g., into
E. coli YI088 containing the plasmid pMC9 (ATCC No. 37195). From about
100,000 recombinant phages there are selected those phages which hybridize
with a suitable probe. Such suitable probes are oligonucleotides which
correspond to a partial sequence of the genomic DNA coding for a
polypeptide in accordance with the invention. The manner in which these
probes are selected and used is well known in the art.
Phages which contain the desired DNA fragment are grown up and the DNA is
isolated. Subsequently, the DNA fragment can be inserted into a suitable
replicable microbial vector. Preferably into an expression vector which
provides the necessary expression signals and which codes for the partial
sequence A of the general formula A--B of the polypeptides of the
invention. The vector pDS78/RBSII,6xHis is a preferred expression vector.
The construction and the production of this vector are described in detail
in the examples. The polypeptides of the present invention can, after
corresponding adaptation of the nucleotide seguence, also be produced in
other suitable expression vectors. Examples of such expression vectors are
described in European patent Application, publication No. 186 069. Other
expression vectors are known to those skilled in the art.
The expression vectors used to make the polypeptides of the invention are
introduced into a suitable host organism. Suitable host organisms are
microorganisms, e.g., yeast cells or bacterial cells which are capable of
expressing polypeptides encoded by the expression vectors. The preferred
host organism is E. coli M15 (described as DZ291 by Villarejo et al. in J.
Bacteriol. 120, 466-474 [1974]). Other suitable host organisms are E.
coli 294 (ATCC No. 31446), E. coli RRl (ATCC No. 31343) and E. coli W3110
(ATCC No. 27325).
The manner in which the expression of the polypeptides of the invention is
carried out depends on the expression vector and on the host organism
used. Usually, the host organisms which contain the expression vector are
grown up under conditions which are optimal for the growth of the host
organism. Towards the end of the exponential growth, when the increase in
the number of cells per unit time decreases, the expression of the
polypeptide is induced, i.e. the DNA coding for the polypeptide is
transcribed and the transcribed mRNA is translated. The induction can be
effected by adding an inducer or a derepressor to the growth medium or by
altering a physical parameter, e.g., by a temperature change. In the
expression vector used in the example below, expression is controlled by
the lac repressor. By adding isopropyl-.beta.-D-thiogalactopyranoside
(IPTG) the expression control sequence is derepressed and thereby the
synthesis of the polypeptide is induced.
The polypeptide produced in the host organisms can be secreted from the
cell by special transport mechanisms or can be isolated by breaking open
the cell. The cell can be broken open by mechanical (Charm et al.. Meth.
Enzymol. 22, 476-556 [1971]). Enzymatic (lysozyme treatment) or chemical
(detergent treatment, urea or guanidine.HCl treatment, etc) means or by a
combination thereof.
In eukaryotes, polypeptides which are secreted from the cell are
synthesized in the form of a precursor molecule. The mature polypeptide
results from cleavage of a so-called signal peptide. As prokaryotic host
organisms are not capable of cleaving eukaryotic signal peptides from
precursor molecules, eukaryotic polypeptides must be expressed directly in
their mature form in prokaryotic host organisms.
The translation start signal AUG, which corresponds to the codon ATG on the
level of the DNA, causes all polypeptides synthesized in a prokaryotic
host organism to have a methionine residue at the N-terminal. In certain
expression systems, this N-terminal methionine residue is cleaved off. It
has, however, been found that the presence or absence of the N-terminal
methionine has no influence on the biological activity of a polypeptide
(see Winnacker, in "Gene und Klone", p. 255, Verlag Chemie, Weinheim, BRD
[1985]).
In cases where the N-terminal methionine is troublesome. It can also be
cleaved off by means of a peptidase which is specific for the N-terminal
methionine. Miller et al. (proc. Natl. Acad. Sci. U.S.A. 84, 2718-2722
[1987] have described the isolation of such a peptidase from Salmonella
typhimurium. The present invention is accordingly concerned with
polypeptides with or without an N-terminal methionine residue.
The polypeptides of this invention can be purified by known methods such as
differential centrifugation. Precipitation with ammonium sulphate,
dialysis (at normal pressure or at reduced pressure). Preparative
isoelectric focusing, preparative gel electrophoresis or various
chromatographic methods such as gel filtration, high performance liquid
chromatography (HPLC), ion exchange chromatography, reverse phase
chromatography and affinity chromatography (e.g., on Sepharose.RTM. blue
CL-6B, on phosphocellulose, on carrier-bound monoclonal anti-bodies
directed against the polypeptide or on metal chelate resins such as those
described in the present invention).
The preferred purification method in the present invention is affinity
chromatographic purification. The purification of the polypeptides on
metal chelate resins (Sulkowsky, Trends in Biotechn. 3, 1-7 [1985]) or on
phosphocellulose is especially preferred. The selective binding cf
neighbouring histidine residues to nitrilotriacetic acid-nickel chelate
resins (NTA resins) and the affinity of aldolases to phosphocellulose are
employed. These two purification methods can also be combined
The polypeptides of the invention can be present in the form of multimers,
e.g., in the form of dimers, trimers, tetramers, or can also be part of
fusion polypeptides Multimers can result when polypeptides are produced in
prokaryotic host organisms, for example by the formation of disulphide
bridges between cysteine residues. Fusion proteins can be produced by
linking DNA fragments which code for a polypeptide of the present
invention with one or more DNA fragments which code for another
polypeptide. Examples of such a fusion polypeptide are polypeptides of the
general formula A--B as defined above. Further examples are polypeptides
of the general formula B--C or A--B--C in which C is another polypeptide
and A and B have the significance given above. Examples of a polypeptide
of the general formula A--B--C are, e.q., fusion polypeptides between part
B in the general formula A--B and .beta.-galactosidase as can be produced
in accordance with Ruther et al., EMBO J., 2, 1791-1794 [1983]. Neither
the affinity peptide A nor the polypeptide C should be detrimental to the
function of the polypeptides as antigens or as vaccines against malaria.
The present invention is also concerned with immunogenic compositions which
contain a polypeptide of the invention and a suitable adjuvant. Suitable
adjuvants for use in human beings and animals are well known in the art
(WHO Techn. Rep. Series 595, 1-40 [1976]; Jollis et al., "Chemical and
Biological Basis of Adjuvants", in Molecular Biochemistry and Biophysics
Vol. 13, 1-148 [1973]. Springer Verlag Berlin).
The polypeptides of this invention can be present as lyophilizates for
reconstitution with sterile water or a salt solution, preferably a saline
solution. By introducing the polypeptides and immunogenic compositions
into mammals, their immune systems are activated to produce antibodies
against the polypeptide. Such antibodies, which are also a part of this
invention, recognize the natural equivalent of the polypeptide in the
malaria parasite and can therefore be used for passive immunization or for
diagnostic purposes.
Antibodies against the polypeptides can be produced in monkeys, rabbits,
horses, goats, guinea pigs, rats, mice, cows, sheep etc.. and also in
human beings. The antiserum or the purified antibodies can be used as
required. The antibodies can be purified in a known manner, e.g., by
precipitation with ammonium sulphate. It is also possible to produce
monoclonal antibodies which are directed against the polypeptides of the
present invention using the method developed by Kohler et al. (Nature,
256, 495-497 [1975]). Polyclonal or monoclonal antibodies can also be used
for the affinity-chromatographic purification of the polypeptides or their
natural equivalents.
The polypeptides and immunogenic compositions of this invention can be used
for the immunization of mammals against malaria. The mode of
administration, the dosage and the number of injections can be optimized
in a manner known to the person skilled in the art. Typically, several
injections are administered over a long time period to obtain a high titre
of antibodies against the malaria antigen.
EXAMPLE
The following abbreviations will be used throughout this example:
______________________________________
ATP adenosine triphosphate
bP base pair
BSA bovine serum albumin
cpm impulse per minute
dATP desoxyadenosine triphosphate
dCTP desoxycytidine triphosphate
dGTP desoxyguanosine triphosphate
dTTP desoxythymidine triphosphate
DTT dithiothreitol
EDTA ethylendiaminetetraacetic acid
IPTG isopropyl .beta.-D-thiogalactopyranoside
kb 1,000 base pairs
kD kilodalton
M molar
mM millimolar
ml milliliter
nm nanometer
PFU plaque-forming units
RPM revolutions per minute
SDS sodium dodecylsulphate
TEMED N,N,N',N'-tetramethylethylenediamine
Tris trishydroxymethane
X-Gal 5-bromo-4-chloro-3-indonyl
.beta.-D-galactopyranoside
______________________________________
Buffers and media
100 .times.Denhardt's (100 ml):
2 g polyvinylpyrrolidone
2 g Ficoll
2 q BSA
100 mg sodium azide
DNA gel loading buffer:
1 .times.TBE (see below for composition)
20% glycerol
0.1% bromophenol blue
0.1% xylenecyanol
Formamide mix:
80% (w/v) formamide
50 mM Tris/boric acid [pH 8.3]
1 mM EDTA
0.1% xylenexyanol
0.1% bromophenol blue
HIN buffer:
10 mM Tris/HCl [pH 7.4]
10 mM magnesium chloride
50 mM sodium chloride
Ligase buffer:
50 mM Tris/HCl [pH 7.8]
10 mM magnesium chloride
20 mM DTT
10 mM dATp
LB medium: per liter:
10 g Bactotrypton
5 g yeast extract
10 g sodium chloride
SDS gel loading buffer:
5% SDS
5mM Tris/HCl [pH 6.8]
200 mM DTT
20% glycerol
0.1% bromophenol blue
20.times.SSC: per liter:
175.3 q sodium chloride
82.2 g sodium citrate [pH 7.0)
SM buffer:
10 mM sodium chloride
10 mM magnesium chloride
10 mM Tris/HCl [pH 7.4]
10.times.T4 polymerase buffer:
0.33 M Tris/acetate [pH 7.9]
0.66 M potassium acetate
0.10 M magnesium acetate
5 mM DTT
1 mg/ml BSA
10 .times.TBE:
0.89 M Tris/boric acid [pH 8.0]
0.89 M boric acid
20 mM EDTA
10 .times.TBS:
0.5 M Tris/HCl [pH 7.4]
1.5 M sodium chloride
100 .times.TE:
1 M Tris/HCI [pH 8.0]
100 mM EDTA
The following methods were used several times in the following example and
are accordingly grouped together here.
Method 1: DNA precipitation with lithium acetate
The DNA solution is treated with a tenth by volume of 5 M lithium acetate
and two volumes of isopropanol, mixed well and placed on dry ice for 10
minutes. The precipitated DNA is centrifuged for 10 minutes at 12,000 RPM
(20.degree. C.) in an Eppendorf bench centrifuge and the supernatant is
carefully removed. The sediment is washed once with 80% (v/v) ethanol and
subsequently dried for 5 minutes in a vacuum centrifuge. The DNA is
dissolved in water and processed further.
Method 2: Agarose Gel Electrophoresis of DNA
The dried DNA is dissolved in 1 .times.DNA gel loading buffer and heated to
65.degree. C. for 5 minutes. 100 ml of 1 .times.TBE buffer are mixed with
agarose (800 mg for a 0.8% gel or 1.2 g for a 1.2% gel) and boiled until
the agarose has dissolved completely. After cooling 2 .mu.l of ethidium
bromide solution (10 mg/ml) are added and the gel solution is poured into
a horizontal gel electrophoresis apparatus (IBI. Genofit. Geneva,
Switzerland). After solidification of the gel the samples are applied to
the gel and the DNA is separated for 2 hours at 150 volt constant voltage.
Commercial standard mixtures of DNA fragments of defined length
(Gibco-BRL. Basle. Switzerland) are used as size markers. The DNA bands
are visualized under 300 nm UV light.
Method 3: Isolation of DNA From an Agarose Gel
The DNA is separated on an agarose gel (Method 2). A piece of NA 45
nitrocellulose membrane (Schleicher and Schuell, Dassel, BRD) is placed in
front of the bands which are to be isolated and the DNA is electrophoresed
on to the membrane for 5 minutes at 200 V. The membrane is removed with
forceps and washed under running. Distilled water. The membrane is
transferred into an Eppendorf test tube and the DNA is eluted at
65.degree. C. for 10 minutes with 200 ul of 1.5 M lithium acetate, 10 mM
Tris/HCl [pH 8.0], 0.1 mM EDTA. The elution is repeated again. The
combined supernatants are treated with 2 volumes of isopropanol. The
precipitated DNA (Method 1) is dissolved in 50 .mu.l of water.
Method 4: Plaque Purification of Lambda Phages
A bacterial culture (e.g. E. coli Y1088) is infected on an agar plate with
lambda phages. Thereby, lytic plaques are formed in the lawn of bacteria.
An agar cylinder (diameter 5 mm) containing a plague is cut from the agar
with a inverted pasteur pipette. The agar cylinder is transferred into an
Eppendorf test tube containing 500 .mu.l of SM buffer and the test tube is
shaken for 5 minutes.
The phage suspension is centrifuged (5 minutes at 12.000 RpM, 20.degree.
C.) and the supernatant is transferred into a fresh test tube. 1 .mu.l of
the phage suspension is diluted with 1 ml of SM buffer, 1, 10 and 100
.mu.l of this solution are added to 50 .mu.l of a cell suspension,
Mg.sup.++ -treated in accordance with Morrison. Methods Enzymol. 68,
326-331 [1979]), of E. coli Y1090 containing the plasmid pMC9 (ATCC No.
37197).
After incubation at room temperature for 30 minutes, the solution is added
to 3 ml of 0.8% (w/v) agar in LB medium and the mixture is poured on to
LB-ampicillin agar plates (LB medium, 40 .mu.g/ml ampicillin). Depending
on the titre, some plates (e.g. those with the 1:1000 dilution) have
individual plaques which, when they are positive in the antibody reaction
or the DNA hybridization, can be isolated. The phages from the plagues can
be grown up and used, e.g., for the isolation of phage DNA.
Method 5: Isolation of Lambda Phage DNA
An individual plaque is picked from an agar plate with a sterile toothpick
and the toothpick is incubated at 37.degree. C. for 30 minutes in 500
.mu.l of SM buffer. The toothpick is removed and the phage solution is
centrifuged (10 minutes at 12,000 RpM. 20.degree. C.). The supernatant is
transferred into a fresh vessel and treated with 50 .mu.l of chloroform.
100 .mu.l of the phage solution are removed and mixed with 50 .mu.l of a
Mg.sup.++ -treated cell suspension of E. coli Y1090 cells (ATCC No.
37197).
After incubation at room temperature for 30 minutes the suspension is mixed
with 3 ml of 0.8% (w/v) agar in LB medium and poured onto LB-ampicillin
agar plates (see Method 4). After incubation at 37.degree. C. for five
hours, the petri dishes are covered with 5 ml of SM buffer and shaken at
room temperature overnight. Thereby, the phages are eluted from the agar.
The SM buffer containing the phages is poured off and centrifuged for 10
minutes at 12,000 RpM (room temperature). The supernatant (phage stock) is
treated with 100 .mu.l of chloroform. The phage titre in the phage stock
usually amounts to 10.sup.10 -10.sup.11 PFU/ml. 50 ml of LB medium
(containing 40 .mu.g/ml of ampicillin and 10 mM magnesium chloride) are
inoculated with 250 .mu.l of a saturated culture of E. coli Y1088
containing plasmid pMC9 (ATCC No. 37195) and 1 ml of phage stock. The
culture is shaken at 37.degree. C. overnight.
After the addition of 2 ml of chloroform cell fragments are centrifuged off
(10 minutes at 12,000 RpM. 20.degree. C.). In each case 50 .mu.l of a
DNase I and RNase solution (in each case 10 mg/ml in water) are pipetted
into the supernatant and the mixture is incubated at 37.degree. C. for 30
minutes. 14 ml of 35% (w/v) polyethylene glycol 6000 (SIGMA, St. Louis,
Mo., USA). 2.5 M sodium chloride are pipetted into the phage suspension
(45 ml) and, after mixing well, the solution is placed on ice for one
hour.
The phages are separated by centrifugation (20 minutes at 12.000 RpM.
4.degree. C.) and dissolved in 1 ml of SM buffer. After the addition of 10
.mu.l of 25% (w/v) lithium dodecylsulphate solution (Serva, Chemie
Brunschwig AG, Basle, Switzerland) and 5 .mu.l of 0.5 M EDTA [pH 8.0] and
a spatula tip of proteinase K (Merck, Darmstadt, BRD) the phage particles
are lyzed at 65.degree. C. for 10 minutes. 1 volume of phenol, which has
previously been saturated with 1 M Tris/HCl [pH 8.0]. is added to the
lysate. After mixing well the phases are separated by centrifugation (5
minutes at 6000 RpM). The supernatant is removed, the extraction is
repeated and the DNA is precipitated from the second supernatant according
to Method 1.
Method 6: Vector Preparation
1 .mu.g of plasmid or phage DNA is digested at 37.degree. C. for 1 hour
with 10 units of restriction enzyme in 100 .mu.l of T4 polymerase buffer.
400 .mu.l of water and 5 units of bacterial phosphatase (Gibco-BRL) are
pipetted in and the DNA is dephosphorylated at 65.degree. C. for one hour.
The solution is extracted twice with phenol and precipitated (Method 1).
The vector fragment is purified via an agarose gel (Method 2), isolated
(Method 3) and dissolved in 50 .mu.l of water.
Method 7: Transformation of E. coli
A 3 ml LB culture is inoculated with E. coli cells and shaken at 37.degree.
C. overnight. 1 ml of this saturated culture is used to inoculate a 50 ml
LB liquid culture. This is sbaken until the optical density at 600 nm
(OD.sub.600) has reached a value of 0.2. The cells are sedimented (5
minutes at 6000 RpM, room temperature) and resuspended o in 50 ml of
ice-cold 50 mM calcium chloride. The solution is placed on ice for 30
minutes. The cells are again centrifuged off (see above) and suspended in
10 ml of 50 mM calcium chloride, 20% glycerol. The competent cells are
frozen at -80.degree. C. in 500 ul portions.
For the transformation, a portion is thawed slowly on ice (30 minutes). 10
.mu.l of DNA solution (1-10 ng), 8 .mu.l of 30% (w/v) polyethylene glycol
(SIGMA), 10 .mu.l of 500 mM magnesium chloride 100 mM calcium chloride and
72 .mu.l of water are mixed well and incubated with 100 .mu.l of competent
cells for 20 minutes on ice. Subsequently, the mixture is incubated at
room temperature for a further 10 minutes.
When using vectors of the M13 type (Yanisch-perron et al., Gene 33, 103-119
[1985]). there are now added 50 .mu.l of 10% (w/v) X-Gal (Gibco-BRL) in
dimethylformamide, 10 .mu.l of 100 mM IPTG (Gibco-BRL) in water and 50
.mu.l of a saturated TG-1 (Amersham, Braunschweig, BRD) culture. After
mixing well 3 ml of 0.8% (w/v) agar in LB medium are added and the mixture
is poured on to a LB agar plate. The Petri dishes are incubated at
37.degree. C. overnight.
When using plasmid DNA, which can be selected for antibiotic resistance
(pUC, pDS78/RBSII, 6xHis, etc.), 1 ml of LB medium is added to the
transformation mixture, and the incubation is carried out at 37.degree. C.
for one hour. The cells are centrifuged off for 3 minutes at 6,000 RPM
(room temperature) and resuspended in 100 .mu.l of LB medium. These 100
.mu.l are distributed uniformly by means of a rotating disc (Schutt,
Gottingen, BRD) on a LB agar plate which contains the antibiotic required
for the selection and likewise incubated at 37.degree. C. overnight.
Method 8: preparation of the DNA for Sequencing
When TG-1 bacteria having a vector of the M13 type which contains a DNA
fragment to be sequenced are transformed as described above, white plaques
result. These white plaques are picked with a toothpick and resuspended in
3 ml of LB medium. Thereto there are added a further 10 .mu.l of a
saturated TG-1 culture. The mixture is shaken at 37.degree. C for 5 hours.
1.5 ml of culture are transferred into an Eppendorf test tube and
centrifuged (5 minutes at 12,000 RpM. 20.degree. C.). 800 .mu.l of
supernatant are transferred into a new test tube and mixed with 200 .mu.l
of 20% (w/v) polyethylene glycol. 2.5 M sodium chloride solution and
incubated at room temperature for 20 minutes. The remainder of the culture
is stored at 4.degree. C. or used for the preparation of "mini-lysate" DNA
(Method 10).
The phages are precipitated by centrifugation (10 minutes at I2.000 RpM,
20.degree. C.). The sediment is dissolved in 100 .mu.l of 1.times. TE
buffer and extracted with 100 .mu.l of saturated phenol. The phases are
separated by centrifugation (5 minutes at 12,000 RpM). 80 .mu.l of the
aqueous phase are transferred into a new reagent test tube, the DNA is
precipitated and dissolved in 12 .mu.l of water (Method 1).
Method 9: DNA Sequencing According to Sanger
3 .mu.l of the DNA prepared according to Method 8 are mixed with 2 .mu.l of
water, 1 .mu.l of HIN buffer. 1 .mu.l of 25 .mu.M dATp, 2 .mu.l of
alpha-[.sup.32 p]-ATp (6000 Ci/mmol, Amersham) and 1 .mu.l of sequencing
starter (pharmacia, Dubendorf, Switzerland) and heated at 55.degree. C.
for 5 minutes. Thereafter, the solution is placed on ice. In the meantime,
4 test tubes each containing 3 .mu.l of the stop solutions A.degree.,
G.degree., T.degree. and C.degree. are prepared. The stop solutions have
the following composition:
A.degree.: 3 .mu.M ddATp, 112 .mu.M dCTp, 112 .mu.M dGTp, 112 .mu.M dTTp
C.degree.: 100 .mu.M ddCTp, 11.5 .mu.M dCTp, 112 .mu.M dGTp, 112 .mu.M dTTp
G.degree.: 100 .mu.M ddGTp, 112 .mu.M dCTp, 5.6 .mu.M dGTp, 112 .mu.M dTTp
T.degree.: 500 .mu.M ddTTp, 85 .mu.M dCTp, 85 .mu.M dGTp, 5.6 .mu.M dTTp.
5 units of Klenow polymerase (pharmacia) are pipetted into the test tubes
containing the DNA and mixed well. In each case 3 .mu.l of this solution
are mixed with the stop solution and incubated at 37.degree. C. for 10
minutes. 1 .mu.l of 0.25 mM dATp is added to each of the four test tubes,
mixed and again incubated for 10 minutes. Finally, the reaction is stopped
by adding 2 .mu.l of formamide mix and heating to 96.degree. C. for 5
minutes. The DNA is now applied to a 0.2 mm gel of the following
composition:
6 ml 10.times. TBE-buffer
28.8 g urea
3.6 ylamide (Bio-Rad Laboratories AG, Glattbrugg, Switzerland)
180 mg bisacrylamide (Bio-Rad)
400 .mu.l 10% ammonium persulphate
20 .mu.l TEMED
The DNA is separated electrophoretically for 1 to 6 hours at 40 watts
constant output. The glass plates are separated and the gel is fixed for 5
minutes in 10% (v/v) acetic acid and 10% (v/v) methanol. The gel is then
washed twice with 10% (v/v) methanol for 5 minutes, mounted on Wharman 3MM
paper (Bender and Hobein, Zurich, Switzerland) and dried in a gel dryer.
The dried gel is autoradiographed for 2 to 20 hours with KODAK-XAR film
(Eastman Kodak Co., Rochester, N.Y., USA).
Method 10: DNA Isolation on a Small Scale ("Mini-Lysate")
About 1 to 2 ml of bacterial culture (e.g., E. coli TG-1 containing a
vector of the M13 type; see Method 8) are centrifuged for 5 minutes at
12,000 RpM (20.degree. C). The supernatant is carefully sucked off. The
sedimented cells are resuspended in 500 .mu.l of 50 mM Tris/HCl [pH 7.6].
5 mM EDTA. After the addition of a small spatula tip of lysozyme (SIGMA)
the suspension is incubated at room temperature for 5 minutes. 15 .mu.l of
25% (w/v) lithium dodecylsulphate solution (SIGMA) and 30 .mu.l of 5 M
potassium acetate are then added and the suspension is mixed carefully.
After incubation on ice for 15 minutes the sample is centrifuged for 15
minutes at 12,000 RpM (4.degree. C). The supernatant is decanted into a
new test tube and treated with 50 .mu.l of RNase solution (10 mg/ml).
After incubation at 37 .degree. C. for 5 minutes, the sample is extracted
once with phenol and once with chloroform (in each case the same volumes).
The DNA in the aqueous phase is precipitated (Method 1) and finally
dissolved in 100 .mu.l of water.
Method 11: Radioactive Labelling of DNA ("Nick Translation")
The following reagents are pipetted into 20 .mu.l of DNA solution:
5 .mu.l of HIN buffer. 10 .mu.l of alpha-[.sup.32 p]-ATp (6000 2 30
Ci/mMol, Amersham), 5 .mu.l of DNase I (1 ng/ml), 5 .mu.l of 1 mM dCTp,
dGTp, dTTp and 5 .mu.l of DNA polymerase I (Boehringer Mannheim AG,
Rotkreuz, Switzerland). The batch (50 .mu.l) is incubated at 14.degree. C.
for 90 minutes and subsequently extracted once with phenol (see Method 5).
The aqueous phase contains the DNA probe and is used directly for
hybridization experiments.
Method 12: Hybridization of DNA
The filter containing DNA is incubated for one hour at 60.degree. C. with
pre-hybridization mix (2 .times. SSC, 0.1% (w/v) lithium dodecylsulphate,
10 .mu.g/ml of denatured calf thymus DNA, 5 .times.Denhardt's, 5 .times.TE
buffer). The calf thymus DNA is previously denatured by boiling. The
pre-hybridization mix is replaced by hybridization mix which corresponds
to the pre-hybridization mix, but which additionally contains about
10.sup.7 cpm of radioactive sample. After incubation at 60.degree. C.
overnight the filters are washed 3 times for 30 minutes in 2 .times.SSC.
The filters are dried and exposed overnight against Kodak XAR film.
Method 13: preparation of a 12% SDS polyacrylamide gel according to
Laemmli, Nature 227, 680-685 [1970]
60 ml separating gel
15 ml 1.5.M Tris/HCl [pH 8.8].
0.4% (w/v) SDS, 8mM EDTA.
24 ml 29% (w/v) acrylamide (Bio-Rad),
1% (w/v) bisacrylamide (Bio-Rad) in water
25 ml water.
500 .mu.l 10% (w/v) ammonium persulphate in water.
The solutions are mixed. Immediately before pouring between 2 glass plates
100 .mu.l of TEMED are added. After the separating gel has polymerized the
collecting gel having the following composition is poured in:
20 ml collecting gel:
5 ml 0.5 M Tris/HCl [pH 6.8].
0.4% (w/v) SDS, 8 mM EDTA.
3 ml 29% (w/V) acrylamide, 1% (w/v) bisacrylamide in water.
12 ml water.
250 .mu.l 10% (w/v) ammonium persulphate solution in water.
After mixing, 30 .mu.l of TEMED are added and a probe comb is inserted
prior to the polymerization. 190 mM glycine. 25 mM Tris [pH 7.6], 1% (w/v)
SDS is used as the electrophoresis buffer. Commercial molecular weight
standards (Bio-Rad) are applied as size markers.
Method 14: Immunoblots (Western blot)
4 .mu.l of a protein sample are separated on a 12% SDS polyacrylamide gel
for 45 minutes al 25 mA constant current. The gel is removed and placed in
transfer buffer. A sheet of nitrocellulose (Schleicher & Schuell).
moistened with water, is placed on the gel. Gel and nitrocellulose are
covered with Whatman 3MM paper and then a sponge is placed on each of
them. The sandwich which is thus obtained is then introduced into an
electrophoresis apparatus, whereby the nitrocellulose is directed towards
the positive pole. The transfer of the proteins is effected at 400 mA
constant current for 15 minutes. After the transfer, the nitrocellulose is
shaken for 10 minutes in 1 .times.TBS buffer.
The nitrocellulose is pre-incubated for 30 minutes in 1 .times.TBS, 5%
(w/v) skimmed milk powder. The antibody against the parasite antigen is
diluted in the ratio 1:1000 in 1 .times.TBS. 5% (w/v) skimmed milk powder
and incubated for one hour with the nitrocellulose sheet. Thereafter, the
sheet is washed five times for three minutes in fresh 1 .times.TBS and
subsequently incubated for one hour with goat-anti-rabbit-peroxidase serum
(Biorad; diluted 1:1000) in 1 .times.TBS, 5% (w/v) skimmed milk powder.
The nitrocellulose is again washed as above and subsequently placed in 5
ml of 1 .times.TBS.
The solution is treated with 10 ml of a 4-chloronaphthol solution (SIGMA,
50 mg/ml in methanol) and mixed well. The colour reaction is started by
the addition of 50 .mu.l of hydrogen peroxide. After the bands have been
made visible, the nitrocellulose sheet is stored in water to prevent an
overexposure. Pre-stained marker proteins, which are used according to
details of the manufacturer (e.g. Gibco-BRL), are employed as the
molecular weight markers.
Method 15: Southern Transfer of DNA onto Nylon Membranes
About 10 .mu.g of plasmid or parasite DNA per trace are separated on an
agarose gel (Method 2). After elution the gel is photographed and agitated
twice for 15 minutes in 0.2 N HCl. Subsequently, the gel is agitated twice
for 15 minutes in 0.5 M sodium hydroxide solution. The gel is neutralized
twice for 15 minutes in 0.5 M Tris/HCl [pH 8.0], 1.5 M sodium chloride,
and placed on a sponge which is soaked with 20 .times.SSC. A nylon
membrane (pALL, Basle, Switzerland) is placed on the gel, followed by 3
sheets of Whatman 3MM paper and about 20 paper towels. The assembly is
weighed down from above with a 500 g weight. After 3 hours the membrane is
removed and dried, firstly at room temperature and subsequently for 1 hour
at 80.degree. C. in a vacuum. The membrane can be treated further in
accordance with Method 12.
Construction of the plasmid pDS78/RBSII,66xHis
1. Description of the plasmids pDS78/RBSII and pDMI,l
The plasmid pDS78/RBSII was used for the construction of the plasmid
pDS78/RBSII,6xHis. E. coli M15 cells transformed with this plasmid and
with the plasmid pDMI.1 have been deposited at the Deutsche Sammlung von
Microorganism in Gottingen on the Sep. 3, 1987[E. coli M15 (pDS78/RBSII;
pDMI,l). DSM No. 4232].
The part of pDS78/RBSlI (FIG. 1 and 2), which lies between the restriction
cleavage sites for Xbal and XhoI and the replication region as well as the
gene for .beta.-lactamase, which confers ampicillin resistance to the
cells, stems originally from the plasmid pBR322 (Bolivar et al., Gene 2,
95-113 [1977]; Sutcliffe, Cold Spring Harbor Symp. Quant. Biol. 43, 77-90
[1979]). However, the gene for the .beta.-lactamase is modified by
elimination of the cleavage sites for the restriction enzymes HincII and
pstI. These alterations in the DNA sequence do not, however, affect the
amino acid sequence of the .beta.-lactamase. The remaining part of the
plasmid carries the regulatable promoter/operator element N250pSN250p29
(R. Gentz, Thesis, University of Heidelberg, BRD [1984]) and the ribosomal
binding site RBSII. This ribosomal binding site was derived from the
ribosomal binding site of the promoter p.sub.G25 of the E. coli phage T5
(R. Gentz. supra) and is obtained as the EcoRI/BamHI fragment by DNA
synthesis. There follows the dihydrofolate reductase gene of the mouse
cell line AT-3000 (Chang et al., Nature 275, 617-624 [1978]; Masters et
al., Gene 21, 59-63[1983]) which has been altered by introducing a
cleavage site for the restriction enzyme BglII directly in front of the
termination codon for translation. Furthermore, the plasmid pDS78/RBSII
contains the terminator t.sub.o of the E. coli phage lambda (Schwarz et
al., Nature 272, 410-414 [1978]), the promoter-free gene of
chloramphenicol acetyltransferase (Marcoli et al., FEBS Letters, 110,
11-14 [1980]) and the terminator Tl of the E. coli rrnB operon (Brosius et
al., J. Mol. Biol., 148, 107-127 [1981]).
pDS78/RBSII contains the regulatable promoter/operator element
N250pSN250p29 and the ribosomal binding site RBSlI. Because of the high
efficiency of this expression signal, the plasmid pDS78/RBSII and its
derivatives such as the plasmid pDS78/RBSII,6xHis can be stably maintained
in E. coli cells only when the promoter/ operator element is repressed by
the bonding of a lac repressor to the operator. The lac repressor is coded
by the lacI gene. N250pSN250p29 can be repressed efficiently only when a
sufficient number of repressor molecules is present in the cells.
Therefore, the lacI.sup.q allele, which contains a promoter mutant leading
to an increased expression of the repressor gene, was used. This
lacI.sup.q allele is contained in the plasmid pDMI,1 (FIG. 3 and 4).
This plasmid carries, in addition to the lacI gene, the neo gene which
confers kanamycin resistance to the bacteria. Kanamycin resistance can be
used as the selection marker. PDMI,1 is compatible with the
above-mentioned plasmids. E. coli cells which are transformed with the
expression vectors described above must contain pDMI,1 to guarantee that
the expression vector is held stable in the cells. An induction of this
system is achieved by adding IpTG to the medium at the desired cell
density.
The plasmid pDMI,1 (FIG. 3 and 4) carries the neo gene of neomycin
phosphotransferase from the transposon Tn5 (Beck et al., Gene 19, 327-336
[1982]). which confers kanamycin resistance to the E. coli cells, and the
lacI gene (Farabough, Nature 274, 765-769 [1978]) with the promoter
mutation I.sup.q (Calos, Nature 274, 762-765 [1978]), which codes for the
lac repressor. Moreover, the plasmid pDMI,1 contains a region of the
plasmid pACYC184 (Chang et al., J. Bacteriol, 134, 1141-1156 [1978]).
which contains all information required for the replication and stable
transmission to the daughter cells.
2. Construction of the plasmid pDS78/RBSII,6xHis
For the construction of the plasmid pDS78/RBSII,6xHis (FIG. 5 and 6), the
EcoRI/BamHI fragment of pDS78/RBSI comprising the ribosomal binding site
RBSII was supplemented with a region coding for six histidines.
For this purpose, two complementary oligonucleotides, the nucleotide
sequences of which are represented in FIG. 5 as a double-stranded DNA
sequence, were first produced simultaneously on a multisynthesis apparatus
(described in European patent Application, publication No. 181 491,
published on the 21.05.85), with controlled pore glass (CpG) being used as
the carrier material (Kiefer et al., Immunol. Meth. 3, 69-83 [1985];
Sproat et al., Tetrahedr. Lett., 24 5771-5774 [1983]; Adams et al., J.
Amer. Chem. Soc., 105, 661-663 [1985]). The lyophilized oligonucleotides
were taken up in water and dissolved at 4.degree. C. for 1 hour. The DNA
concentration amounted to 100 nMoles/ml. For the phosphorylation, in each
case 150 pMoles cf the two oligonucleotides in 20 .mu.l of 50 mM Tris/HCl
[pH 8.5]and 10 mM MgCl.sub.2 were incubated at 37.degree. C. for 20
minutes with 2 pMoles of
.gamma.-[.sup.32 p]-ATp (5,000 Ci/mMole, Amersham,) and 1 unit (U) of
T4-polynucleot1de k1nase (Gibco-BRL). Subsequently, 5 nMoles of ATp were
added and, after a further 20 minutes at 37.degree. C, the reactions were
terminated by heating to 65.degree. C.
The DNA of the plasmid pDS78/RBSII was prepared for ligation with the two
phosphorylated oligonucleotides by first cleaving 2 pMoles of the plasmid
DNA with the restriction enzyme BamHI, following the manufacturer's
instructions. The DNA was extracted with phenol, washed with ether and
precipitated as described in Method 1. The sediment was dried and taken up
in 20 .mu.l of TE buffer.
For the ligation with the phosphorylated oligonucleotides, 1.5 pMoles of
the plasmid DNA cleaved with BamHl were incubated at 15.degree. C. for 2
hours with in each case 60 pMoles of the phosphorylated oligonucleotides
in ligase buffer containing 2 units of T4-DNA ligase. After a further
incubation at 65.degree. C. for 5 minutes, the ligated DNA was cleaved
with the restriction enzymes XhoI and BamH according to details of the
manufacturers, before the XhoI/BamHI fragment F comprising the regulatable
promoter N250pSN250p29, the ribosomal binding site RBSII and the region
coding for 6 histidines (FIG. 5) was isolated using Method 3.
For the construction of the plasmid pDS78/RBSII,6xHis, the XhoI/BamHI
fragment F described above was integrated into the plasmid pDS78/RBSII,
whereby the original XhoI/BamHI fragment of this plasmid was replaced
(FIG. 6). For this purpose, 1 pMole of DNA of the plasmid pDS78/RBSII was
first cleaved with the restriction enzymes XhoI and BamHI, and the larger
DNA fragment was isolated using Method 3. 0.05 pMoles of this fragment
were incubated at 15.degree. C. for 2 hours with 0.1 pMoles of the
isolated XhoI/BamHI fragment F in ligation buffer with 2 units of T4-DNA
ligase. E. coli M15 cells transformed with plasmid pDMI,1 were prepared
for the transformation with pDS78/RBSI,6xHis according to the method of
Morrison (Methods Enzymol. 68, 326-331 [1979]).
After heating to 65.degree. C. for 7 minutes, the ligation mixture was
added to 200 .mu.l of these competent cells. The sample was held in ice
for 30 minutes, then incubated at 42.degree. C. for 2 minutes and, after
the addition of 0.5 ml of LB medium, incubated at 37.degree. C. for 90
minutes. The cells were plated out on LB agar plates which contained 100
.mu.g/ml of ampicillin and 25 .mu.l/ml of kanamycin and incubated at
37.degree. C. overnight in an incubator.
Individual colonies were picked with a sterile toothpick and incubated in
10 ml of LB medium containing 10.mu.l/ml ampicillin and 25 .mu.g/ml
kanamycin for 12 hours in a shaking incubator. Thereafter, the cells were
sedimented and the plasmid DNA was isolated according to the method of
Birnboim et al. (Nucleic Acids Res. 7, 1515-1523 [1979]).
In each case 0.2 .mu.g of the isolated plasmids were cleaved with the
restriction enzymes XhoI and BamHI, to examine whether the XhoI/BamHI
fragment F was present in these plasmids. Plasmids having such a fragment
received the designation pDS78/RBSII,6xHis (FIG. 6).
To demonstrate that the correct sequence was present in pDS78/RBSIl,6xHis,
the double-stranded ciroular plasmid DNA was sequenced, with a
.gamma.-[.sup.32 p]-ATp labelled starter sequence ("primer") being used.
This starter sequence contained the nucleotides of position 89-108 of the
plasmid pDS7B/RBSII. 0.3 pMoles of the isolated plasmid DNA were
precipitated with alcohol, and the sediment was washed once with 80% (v/v)
ethanol, dried and dissolved in 8 .mu.l of 1/4 TE buffer.
The sample was incubated at 95.degree. C. for 5 minutes, cooled to
0.degree. C. and centrifuged (2 minutes, 12,000 RpM). 1.5 pMoles of the
starter sequence in a volume of 2 .mu.l were added before the sample was
incubated at 95.degree. C. for 2 minutes and then at 42.degree. C. for 5
minutes. The DNA was then sequenced according to the method of Sanger et
al. (proc. Natl. Acad. Sci. USA 74, 5463-6567 [1977]. Because a
radioactively labelled "primer" was used, all reactions were carried out
with unlabelled deoxynucleotide triphosphates. The DNA sequence analysis
indicated that pDS78/RBSIl,6xHis contained the sequence given in FIG. 5.
Isolation of a p. falciparum gene with antibodies from a genomic expression
gene bank
Construction of the expression gene bank of p. falciparum
p. falciparum cells (Kl isolate) were grown by usual methods (Trager et
al., Science 193, 673-675 [1976]) in 10 culture dishes and subsequently
washed in culture medium containing 0.1% saponin. The washed parasites
were resuspended in 2 ml of 10 mM EDTA, pH 8.0, 0.5% (w/v) SDS. After the
addition of 50 mg of proteinase K (Merck), the mixture was incubated at
65.degree. C. for 10 minutes and then treated with 2 ml of phenol
(saturated with Tris/HCI [pH 8.0]). The phases were mixed by shaking and
again separated by centrifugation (10 minutes at 6,000 RpM, 20.degree. C..
The phenol extraction was repeated twice (an interphase should no longer
be visible).
The DNA was precipitated according to method 1, washed with ethanol, dried
and then dissolved in 2 ml of water and cut mechanically, i.e. squeezed 80
times through a syringe having a 0.5.times.16 mm needle. Thereafter, 0.2
volumes of 5 .times.EcoRl methylase buffer (50 mM Tris/HCl [pH 7.5], 0.25
M NaCl, 50 mM EDTA, 25 mM .beta.-mercaptoethanol, 0.4 mM
S-adenosylmethionine) were added. 10 .mu.g of DNA were methylated at
37.degree. C. for 30 minutes with 50 units of EcoRl methylase (New England
Biolabs. Beverly, Mass., USA).
The DNA was extracted once with phenol as described above and precipitated
in accordance with Method 1. The DNA was dissolved in 200 .mu.l of T4
polymerase buffer and, after the addition of 5 .mu.l of 5 mM dATp, dCTp,
dGTp and dTTp and 10 units of T4 polymerase (Gibco-BRL), incubated at
37.degree. C. for 30 minutes. The DNA was again extracted with phenol and
precipitated in accordance with Method 1.
The DNA was dissolved in 50 .mu.l of T4-DNA ligase buffer and, after the
addition of 0.01 OD260-units of phosphorylated EcoRI oligonucleotide
adaptors (New England Biolabs) and 2 .mu.l of T4-DNA ligase (12 Weiss
units, New England Biolabs), ligated at 14.degree. C. overnight. The DNA
was precipitated according to Method 1. dissolved in 20 .mu.l of 1
.times.DNA gel loading buffer and separated on a 0.8% agarose gel (Method
2). DNA fragments having a length of 2 to 6 kb (1 kb=1,000 nucleotides)
were isolated in accordance with Method 3.
The DNA obtained was dissolved in 50 .mu.l of water and, after the addition
of 6 .mu.l of 10 .times.ligase buffer, 2 .mu.l of dephosphorylated lambda
arms (promega Biotech., Madison, Wis.) and 6 Weiss units of T4-DNA ligase,
ligated at 1.degree. C. overnight. The DNA was precipitated (Method 1) and
dissolved in 5 .mu.l of water. After the addition of 2.mu.l of "packaging
Extract" (Genofit, S.A., Geneva, Switzerland), the DNA was packed in phage
particles at 20.degree. C. for 2 hours according to the directicns of the
manufacturer. After the addition of 500 .mu.l of SM buffer and 50.mu.l of
chloroform, the gene bank was ready for the antibody test.
Antibody test of the gene bank
Antibodies against surface proteins of the merozoite stage of p. falciparum
were produced in rabbits as described by perrin et al. (J. Clin. Invest.
75, 1718-1721 [1984]). An antiserum which was specific for a p. falciparum
K12 merozoite surface antigen having a molecular weight of 41,000 was
selected for the antibody test of the gene bank.
E. coli Y1090 in 3 ml of LB medium containing 40 .mu./ml of ampicillin was
incubated at 37.degree. C. overnight in a shaking bath. On the next
morning the cells were sedimented (10 minutes at 7,000 .times.g.
20.degree. C.) and resuspended in 1 ml of SM buffer. 10.sup.6 infectious
phage particles of the gene bank were added to this cell suspension and
incubation was carried out at room temperature for 30 minutes. 60 ml of
0.8% agar solution in LB medium. warmed to 42.degree. C., were added and
mixed well. The soft agar containing the infected cells was distributed on
6 LB agar plates (diameter 135 mm) containing 40 .mu.g/ml ampicillin and
incubated at 42.degree. C. for 5 hours.
A nitrocellulose filter (Schleicher and Schuell), immersed in 100 mM IpTG
solution and dried, was placed on each dish and incubation was carried out
at 37.degree. C. overnight. On the next day the position of the filter
relative to the dish was marked and the marked filter was stored in 1
.times.TBS. A new nitrocellulose filter, treated in 100 mM IpTG solution,
was placed on the plate, marked and incubated at 37.degree. C. for 4 hours
on the plate. The two filter batches were shaken for 10 minutes in 1
.times.TBS, then incubated for 20 minutes in 1 .times.TBS, 20% FCS (fetal
calf serum).
The rabbit antiserum was diluted 1:1000 with 1 .times.TBS/20% FCS, and the
two filter batches were incubated at room temperature for one hour in a
shaking bath. The filters were washed three times for three minutes in 1
.times.TBS. 0.1% Triton.TM.-X-100 (Bio-Rad) in a shaking bath, followed by
an incubation for one hour with 5 .mu.Ci of [.sup.125 I]-protein A
(Amersham Catalogue No. 1M.144) in 1 .times.TBS, 0.1% protease-free bovine
serum albumin (SIGMA). The filters were again washed as above and the
filters were dried at room temperature.
The filters were exposed overnight against Kodak XAR. plaques which were
present on both plates were identified with the aid of the markings and
picked from the Petri dishes on the basis of the marking. The individual
samples were again plated out in soft agar in different dilutions
according to Method 4, and positive plaques were again identified as
described above. An individual, positive plaque (Kl-A) was picked, the
lambda phages were grown up according to Method 5 and the DNA was
isolated.
10 .mu.g of Kl-A DNA were dissolved with 490 .mu.l of T4 polymerase buffer
and digested at 37.degree. C. for one hour with 50 units of HindIII. The
DNA was precipitated (Method 1) and analyzed on a 0.8% agarose gel (Method
2). 10 .mu.g of gtll DNA were digested with HindIII and analyzed as the
control. A HindIII fragment (270 base pairs) was now present in the trace
having the Kl-A DNA. The fragment was isolated (Method 3). dissolved in 50
.mu.l of water and stored at 4.degree. C.
For sequencing, 50 ng of HindIII-cleaved, dephosphorylated M13 mp18 DNA
(pharmacia; Method 6) were mixed with 10 .mu.l of the dissolved HindIII
fragment from Kl-A, 2 .mu.l of 10 .times.ligase buffer, 6 .mu.l of water
and 6 Weiss units of T4-DNA ligase (New England Biolabs) were added, and
the DNA's were ligated at room temperature for one hour. Competent TG-1 E.
coli cells were transformed with the ligated DNA (Method 7). A white
plaque was isolated, amplified and sufficient DNA for the sequence
determination was isolated (Method 8). The DNA sequence was determined
according to Method 9. An M13 mp18 clone having the HindIII fragment was
denoted as Kl-A-M.
The HindIII fragment from the Kl-A-M DNA was used to isolate a longer piece
of DNA which coded for the merozoite antigen. For this purpose, the
double-stranded DNA of the M13 clone Kl-A-M Was isolated (Method 10).
After the addition of 20 units of HindIII, 5 .mu.g of DNA were digested at
37.degree. C. for one hour. The solution was again precipitated (Method
1). The DNA was separated on a 1.2% agarose gel (Method 2). The 270 bp
HindIII fragment was isolated (Method 3) and the purified DNA was
dissolved in 20 .mu.l of water. The DNA was labelled by "nick translation"
(Method 11).
The p. falciparum lambda gene bank was again plated as described above
(2.times.10.sup.5 phage particles on two Petri dishes of 135 mm diameter).
After five hours, as plagues became visible, the Petri dishes were removed
from the 37.degree. C. incubator and stored overnight in a refrigerator.
pALL nylon filters (pALL. Basle, Switzerland) were placed on the cold
dishes and the relative positions of the filters to the Petri dishes were
marked with ink.
After 5 minutes the filters were withdrawn carefully from the plates and
placed with the side having the plaques upwards on Whatman 3MM paper which
had previously been soaked with alkaline solution (0.5 M sodium hydroxide
and 0.5 M Tris). After a few minutes, the filters were placed on a new
Whatman 3MM paper soaked with the alkaline solution. Thereafter, the
filters were dried briefly on a 3MM filter paper and then placed twice for
five minutes on Whatman 3MM paper which had previously been soaked with
1.5 M NaCl, 0.5 M Tris/HCl [pH 8.0]. The filters were then dried in air
and baked at 80.degree. C. for 90 minutes in a vacuum. The hybridization
of the filters with the 270 bp HindII fragment (1.times.10.sup.7 cpm) as
the probe was carried out according to Method 12.
positive plagues were picked as described above, and the specificity of the
hybridization was examined with the radioactive probe according to Methods
4 and 12. An individual plaque, Kl-B. was grown up according to Method 5
and the DNA was digested with HindIII as described above and separated on
agarose gels (Method 2). The HindIII fragments, which were not present in
the vector DNA, were isolated, cloned in M13 mp18 and sequenced (Methods
6, 7, 8 and 9).
Restriction analysis of the p. falciparum DNA in the Kl-B DNA
1 .mu.g of Kl-B lambda DNA in 50 .mu.l of T4 polymerase buffer was digested
with 5 units of EcoRI for one hour. The DNA was analyzed (Method 2) and a
1.3 kb fragment was isolated (Method 3). The isolated fragment DNA was
dissolved in 20 .mu.l of water. 1 .mu.g of pUC18 DNA (pharmacia) was
digested with 5 units of EcoRI and processed further according to Method
6. After isolation from the gel (Method 3) the linearized vector was
dissolved in 50 .mu.l of water. 1 .mu.l of vector were incubated at room
temperature for 1 hour with 5 .mu.l of 1.3 kb fragment, 1 .mu.l of
10.times.ligase buffer and 6 Weiss units of T4-DNA ligase.
E. coli cells were transformed with the DNA in accordance with Method 7.
and the plasmid DNA was isolated from the transformants (Method 10). The
plasmid obtained was designated as pKI-B. In each case 0.5 .mu.g of pKl-B
DNA were digested as described above with the restriction enzymes SphI,
XmnI, HpaI and, in double digestions additionally with HindIII and
analyzed on an agarose gel according to Method 2. The entire sequence of
the p. falciparum DNA in pKl-B could be determined with the aid of this
restriction analysis.
Expression of the HpaI/SphI fragment in E. coli
A HpaI/SphI fragment specific for p. falciparum was isolated from the clone
pKl-B in accordance with Methods 1 to 3. 6 .mu.g of pKl-B DNA were
digested at 37.degree. C. for one hour with 15 units of HpaI and 15 units
of SphI in 100 .mu.l of 1.times. T4 polymerase buffer. The DNA was
precipitated (Method 1) and separated on a 0.8% agarose gel (Method 2),
and a 700 bp fragment was isolated (Method 3). The fragment was
resuspended in 20 .mu.l of water and, after the addition of 10 pMoles of a
phosphorylated BamHI oligonucleotide adaptor (12-mer: CCCGGATCCGGG; New
England Biolabs). 2.5 .mu.l of 10 .times. ligase buffer and 6 Weiss units
of T4 DNA ligase, ligated at 14.degree. C. overnight. The DNA was
precipitated (Method 1). dissolved in 50 .mu.l of 1.times.T4 polymerase
buffer and, after the addition of 40 units of BamHI, digested at
37.degree. C. for 1 hour.
The DNA was precipitated (Method 1) and separated on a 1.0 percentage
agarose gel (Method 2). A 700 bp fragment was isolated (Method 3) and
dissolved in 10 .mu.l of water. For the preparation of the vector (see
Method 6). 1 .mu.g of pDS78/RBSII,6xHis vector DNA was digested at
37.degree. C. with IO 10 units of BamHI for one hour in T4 polymerase
buffer. The vector DNA was dephosphorylated (Method 4). extracted once
with phenol (see above), purified on a 0.8% agarose gel (Method 2) and
subsequently isolated according to Method 3. The isolated DNA was
dissolved in 50 .mu.l of water
5 .mu.l of the linearized pDS78/RBSII,6xHis vector DNA, which had been
digested with BamHI and dephosphorylated (Method 6). were incubated at
room temperature for one hour with 5 .mu.l of the 700 bp fragment, 1.2
.mu.l of 10.times.ligase buffer and 6 Weiss units of T4-DNA ligase. 10
.mu.l of DNA were then transformed into competent M15 (pDMI,l) cells
(Method 7) and plated on LB plates with 100 .mu.g/ml ampicillin and 25
.mu.g/ml kanamycin. Individual colonies were picked with a toothpick and
transferred into 3 ml of LB medium containing 100 .mu.g/ml ampicillin and
25 .mu.l/ml kanamycin. The cultures were incubated at 37.degree. C. in a
shaking water bath until the optical density at 600 nm (OD.sub.600)
increased to 0.6 compared with pure medium.
An aliquot of 500 .mu.l of the culture was removed as a non-induced
control. IpTG (1 mM final concentration) was added to the remainder of the
culture and the induccd culture was incubated for a further 3 hours.
Thereafter, 500 .mu.l of the induced culture were removed and centrifuged
together with the non-induced sample (3 minutes at 12,000 RpM, 20.degree.
C.). The supernatant was sucked off and the cell sediment was resuspended
in 100 .mu.l of SDS sample buffer.
The samples were boiled for 7 minutes and the proteins were separated on a
12% SDS gel (Method 13) by means of electrophoresis (three hours at 50 mA
constant current). The gel was stained for 30 minutes on the shaker with
0.1% Coomassie blue in 30% (v/v) acetic acid and 10% (v/v) methanol. The
gel was decolorized at 65.degree. C. for 2 hours in 10% (v/v) methanol and
10% (v/v) acetic acid. Clones which, compared with the uninduced sample,
exhibited an additional band having the expected molecular weight of 27 kD
were analyzed according to Method 14. The novel protein was denoted as
protein (27 kD). The amino acid sequence of the expressed protein
corresponded to amino acid sequence (II).
Analysis of 11 different parasite isolates with antibodies against the
antigen
The following 11 isolates of p. falciparum were tested: RO-33, Ghana;
RO-56, Ethiopia; Geneva No. 13, Senegal; H-B3, Honduras; RO-53, Cameroon;
R-FCR 3, Gambia; MAD-20, papua New Guinea; 542, Brazil; RO-58, East
Africa; FCH-5-C2, Tanzania; Kl, Thailand. The parasites were isolated from
malaria patients and cultivated according to standard methods. Malaria
parasites from other isolates of p. falciparum can, however, also be used.
Parasites from two culture dishes were centrifuged off (10 minutes at
1,500 RpM, 4.degree. C.) and dissolved in 200 .mu.l of SDS-gel loading
buffer. After boiling for seven minutes the samples were separated on a
SDS gel (Method 13). The samples were transferred to nitrocellulose and
tested with antibodies against the antigen (Method 14). The result (FIG.
7) showed that the antigen was present in all isolates and had a similar
molecular weight in all isolates.
Analysis of parasite isolates with gene probes from Kl
The EcoRI fragment from pKl-B was isolated in accordance with Methods 2 and
3 and labelled by "nick translation" (Method 11). The labelled probe was
used as the hybridization probe (10.sup.6 cpm). In each case 10 .mu.g of
DNA were isolated (see above) from different p. falciparum isolates,
digested with 50 units of DraI in T4 polymerase buffer and separated on a
1.2% agarose gel (Method 2). The DNA was transferred to a nylon filter
(Method 15) and subsequently hybridized (Method 12). The result (FIG. 8)
showed a uniform band pattern in the case of all tested isolates and
proved together with sequence data of DNA from different isolates on the
plane of the DNA that the antigen, which corresponds to the polypeptides
of the invention, is preserved.
Expression and purification of protein (41 kD)
1. Construction of the expression vector
1 .mu.l of pKI-B DNA (concentration 0.5 .mu.g/.mu.l) was mixed with 100
.mu.l of 1 .times.T4 polymerase buffer. After the addition of 5 units of
EcoRI the mixture was incubated at 37.degree. C. for 1 hour. The sample
was precipitated with isopropanol (Method 1) and separated on a 0.8%
agarose gel (Method 2). The 1.3 kb EcoRI fragment was isolated according
to Method 3. 0.5 .mu.g of M13 mp 18 DNA (pharmacia) was incubated with
EcoRI and phosphatase (Method 6). 5 .mu.l of vector DNA were mixed with 5
.mu.l of EcoRI fragment from pKl-B, 2 .mu.l of 10 .times.ligase buffer, 7
.mu.l of water and 1 .mu.l of T4-DNA ligase (6 Weiss units, pharmacia) and
ligated at 14.degree. C. overnight.
The DNA obtained was transformed into E. coli TG-1 according to Method 7. A
white plaque was picked (Method 8) and used to inoculate 20 ml of LB
medium which had been treated with 200 .mu.l of a saturated TG-1 culture.
The culture was shaken at 37.degree. C. for 5 hours and the cells were
subsequently centrifuged for 5 minutes at 12,000 RpM and 4.degree. C. The
cells were washed once in water and again centrifuged.
The M13 DNA (MKl-B) was isolated according to Method 10. 50 .mu.l of DNA
were digested at 37.degree. C. for 1 hour with 5 units each of pst I and
BamHI and precipitated according to Method 1. The DNA sediment was
dissolved in 100 .mu.l of exonuclease III buffer (66 mM Tris/HCl [pH 8.0]
6.6 mM MgCl.sub.2) After the addition of 10 .mu.l of exonuclease III
(Gibco-BRL, 5000 units/77 .mu.l) the mixture was incubated ar room
temperature for 30 seconds.
After the addition of 10 .mu.l of 0.5 M EDTA the sample was inactivated at
65.degree. C. for 10 minutes and precipitated according to method 1. The
sediment was dissolved in 50 .mu.l of Sl buffer (2 mM potassium acetate. 1
mM zinc sulphate, 5% (w/v) glycerol) and, after the addition of 10 units
of Sl nuclease (Giboo-BRL), incubated at room temperature for 30 minutes.
The sample was extracted twice with phenol (Method 6) and the DNA was
precipitated (Method 1). The DNA was dissolved in 12 .mu.l of HIN buffer.
After the addition of 1 .mu.l of Klenow polymerase (5 units, pharmacia)
the mixture was incubated at room temperature for 2 minutes and, after the
addition of 1 .mu.l of 2 mM dATp, dCTp, dGTp, dTTp, again incubated at
37.degree. C for 2 minutes. 30 .mu.l of water, 5 .mu.l of 10 x ligase
buffer and 1 .mu.l of T4-DNA ligase (6 Weiss units, pharmacia) were added
and the batch was ligated at 14.degree. C. overnight.
The mixture was transformed into E. coli TG-1 (Method 7). 4 white plaques
were picked, the DNA was prepared (Method 8) and analyzed by sequencing
(Method 9). The DNA used for the expression (see below) was named M2/13.
50 .mu.l of M2/13 DNA were digested completely with 20 units of EcoRI. The
DNA was precipitated and dissolved in 50 .mu.l of T4 polymerase buffer.
The DNA was digested partially at 37.degree. C. for 2 minutes with 1 unit
of HindIII, precipitated (Method 1) and separated on a 0.8% agarose gel
(Method 2).
The 1.3 kb DNA EcoRI-HindIII fragment was isolated (Method 3). 50 .mu.l of
DNA solution. 6 .mu.l of 10 .times.HIN buffer, 1 .mu.l of Klenow
polymerase (5 units. Pharmacia) and 2 .mu.l of 5 mM dATp, dCTp, dGTp, dTTp
were mixed and incubated at 37.degree. C. for 30 minutes. The DNA was
precipitated (Method 1). resuspended in 10 .mu.l of water and, after the
addition of 10 pMoles of a phosphorylated BamHI oligonucleotide adaptor
(8-mer: CGGATCCG; New England Biolabs) as well as 2.5 .mu.l of 10
.times.ligase buffer and 6 Weiss units of T4-DNA ligase, ligated at
14.degree. C. overnight. The DNA was precipitated (Method 1) and separated
on a 1.0% aragose gel (Method 2). The 1.3 kb DNA fragment was isolated and
ligated with 0.1 .mu.g of pDS78/RBSII,6xHis vector as described above. The
new plasmid obtained was denoted at p8/3. The nucleotide sequence of this
plasmid is shown in FIG. 11.
Further analysis of the clones containing plasmid p8/3 was carried out as
described above by SDS protein gels (Method 13) and immunoblots (Method
14). The polypeptide expressed from plasmid p8/3 is a protein having a
molecular weight of about 41,000 Dalton. The protein, which is denoted
hereinafter as (41 kD), was purified as follows.
2. purification of the protein (41 kD) from E. coli
60 g of recombinant E. coli cells containing p8/3 were disintegrated in two
portions each of 30 g for three 1-minute periods in a cell homogenizer
(model MSK, Braun, Melsungen, BRD) with in each case 70 g of glass
grinding elements (diameter 0 1 mm) and in each case 10 ml of buffer A (50
mM Tris/HCl [pH 7.0], 50 mM KCl). The cell material was diluted with 150
ml of buffer A and centrifuqed (10.000 .times.g. 30 minutes. 4.degree.
C.). The desired protein (41 kD) remained in dissolved form in the
supernatant (crude extract).
20 g of Cellex p (Bio-Rad) were soaked in buffer A and packed into a column
(diameter =5 cm, length .TM.7 cm). After equilibration of the column with
buffer A, the crude extract was applied to the column with a pump
(throughflow rate =170 ml/hr.). The adsorbed protein (Cellex p eluate) was
eluted by increasing the phosphate concentration (gradient with 1 M
potassium phosphate [pH 7.0]).
Thereafter, the Cellex p eluate was adsorbed on a nickel-nitrilotriacetic
acid resin which was produced according to Hochuli et al., J. Chromatogr.
411, 177-184 [1987]. The NTA resin column (diameter =1.6 cm, length =9 cm,
throughflow rate =170 ml/hr.) was equilibrated with 0.1 M Tris/HCl [pH
7.5], 0.5 M NaCl. and the adsorbed protein was eluted (NTA eluate) by
means of a gradient of 0 to 0.5 M imidazole.
The NTA eluate was concentrated by means of ultrafiltration on a YMIO
membrane (Amicon, Div. W.R. Grace & Co., Danvers, Mass., USA) and
chromatographed on a Sephacryl.RTM. S-200 column (pharmacia, diameter =2.6
cm, length =83 cm, throughflow rate =14.6 ml/hr.) in pBS buffer (80 g
NaCl, 2 g KCl, 2 g KH.sub.2 pO.sub.4, 29 g Na.sub.2 HpO.sub.4 .multidot.12
H.sub.2 O in 10 1 of pyrogen-free water). The yield of purified protein
was 9 mg (determined according to Lowry, J. Biol. Chem. 193, 265-275[1951]
with BSA as the standard).
3. Immunological and biochemical analysis of the protein
The protein (41 kD), purified as described above, was analyzed by means of
polyacrylamide gel electrophoresis and Western blot (Towbin et al., supra)
(FIG. 10). Trace 3 (FIG. 10a) shows that the (41 kD) protein was greatly
enriched in comparison to the E. coli proteins. In the final product
(trace 5), E. coli proteins were no longer visible (FIG. 10b). FIG. 10c
shows that the purified protein (41 kD) was present partially as a
homodimer. This homodimer formed spontaneously. It could be separated into
monomers only partially by treatment with mercaptoethanol.
The amino acid sequence of the protein (41 kD) expressed from plasmid p8/3
corresponded to amino acid sequence (III"').
By comparing the amino acid seguence of protein (41 kD) with the amino acid
sequence of known proteins, it was established that the protein (41 kD)
had a strong homology to aldolases. Investigations were therefore carried
out to determine whether the purified protein had aldolase activity.
An aldolase colour test was used according to the manufacturer's
instructions (SIGMA). The purified protein (41 kD) exhibited a specific
activity of 13 .mu.Moles of fructose-1,6-diphosphate per mg of protein per
minute at 37.degree. C.
The endotoxin content of the purified protein (41 kD) was determined by
means of a LAL test (pyroquant Diagnostik GmbH, BRD) according to the
manufacturer's instructions. An endotoxin content of less than 3.1 EU/mg
of protein was measured (EU .TM.endotoxin units).
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